Analysis of Biochemical and Antimicrobial Properties of Bioactive Molecules of Argemone mexicana

This study identified phytochemicals in Argemone mexicana (A. mexicana) extracts that are responsible for its medicinal properties, and the best solvent for their extraction. The extracts of the stem, leaves, flowers, and fruits of A. mexicana were prepared at low (corresponding to room temperature) and high temperatures (corresponding to the boiling points) in various solvents, viz., hexane, ethyl acetate, methanol, and H2O. The UV-visible absorption spectra of various phytoconstituents in the isolated extracts were determined through spectrophotometry. Qualitative tests for the screening of phytoconstituents in the extracts were performed to identify various phytochemicals. We identified the presence of terpenoids, alkaloids, cardiac glycosides, and carbohydrates in the plant extracts. The antioxidant and anti-human immunodeficiency virus type 1 reverse transcriptase (anti-HIV-1RT) potential, as well as the antibacterial activity of various A. mexicana extracts were determined. These extracts showed strong antioxidant activities. The extracts exhibited antimicrobial activities against Salmonella typhi, Staphylococcus epidermis, Citrobacter, Neisseria gonorrhoeae, and Shigella flexineri. These extracts significantly inhibited HIV-1 reverse transcriptase activity. The aqueous leaf extract prepared at a temperature equivalent to the boiling point, i.e., 100 °C, was identified to be the most active against pathogenic bacteria and HIV-1 RT.


Introduction
Argemone mexicana L. (A. mexicana) (Papaveraceae), also known as the Mexican prickly poppy or Mexican poppy, grows in the tropical and subtropical regions globally. Argemone mexicana L. is mainly found in Mexico but it is now widely distributed across many parts of the world, including India, Bangladesh, the United States and Ethiopia [1,2]. A. mexicana, a globally used medicinal plant, serves as a source of many alternative medicines. The herbs/shrubs and many trees present in the wild state possess a huge number of novel phytochemicals of medicinal significance. Studies on the wild species of certain weeds have been considered of great importance for the treatment of various diseases [3].

Phytochemical Analysis of Phytochemicals from A. mexicana Extracts at Room Temperature as Detected by TLC
To confirm the results obtained from spectral analysis, TLC of the fractions which showed the presence of optimal concentrations of phytochemicals was carried out. These results are presented in Figure 1. The ethyl acetate fruit extract of A. mexicana showed the presence of alkaloids, flavonoids, primary and secondary amines. Other phytochemicals included antioxidants, carbohydrates, saponins, glycosides, essential oil, phenols, mycotoxin, and terpenes. rence of phytochemicals. In the leaf extract produced at a high temperature in hexan ethyl acetate, methanol, and aqueous medium, the most abundant compounds we flavonoids, with absorption peaks observed at 231, 236, 405, 666, and 668 nm. Pheophyt A (absorbance peaks at 237, 376, and 669 nm) and the unsaturated carbonyl compound (peak at 205 nm) were also observed.
The stem and fruit prepared at a high temperature in hexane, ethyl acetate, meth nol, and aqueous medium extracts contained flavonoids, with absorption peaks noted 227, 350, 380, 666, and 669 nm. Pheophytin A was the second most abundant phyt chemical in the stem extract across all the solvents; the peak was noted at 422 nm. Phe phytin A was not present in the methanolic extract of the fruit, and the extracts of ste and fruit prepared in water. The unsaturated carbonyl compounds were found in th aqueous stem extract at 208 nm only. In the flower extract, flavonoids and pheophytin were identified (239, 351, and 669 nm) in the methanolic extract, and unsaturated ca bonyl compounds at 210 nm at lower solvent concentrations (Supplementary Figures an Tables).

Phytochemical Analysis of Phytochemicals from A. mexicana Extracts at Room Temperatur as Detected by TLC
To confirm the results obtained from spectral analysis, TLC of the fractions whic showed the presence of optimal concentrations of phytochemicals was carried out. The results are presented in Figure 1. The ethyl acetate fruit extract of A. mexicana showed th presence of alkaloids, flavonoids, primary and secondary amines. Other phytochemica included antioxidants, carbohydrates, saponins, glycosides, essential oil, phenols, m cotoxin, and terpenes.

Phytochemical Analysis of A. mexicana Extracts Prepared at the Boiling Points of Solvents (High Temperature) Using TLC
The results are shown in Figure 2. The extract of A. mexicana leaves produced in water at 100 • C indicated the occurrence of flavonoids and the primary/secondary amines. Antioxidants, glycosides, steroids, carbohydrates, prostaglandins, saponins, essential oil, phenols, and terpenoids were also detected. The results are shown in Figure 2. The extract of A. mexicana leaves produced in water at 100 °C indicated the occurrence of flavonoids and the primary/secondary amines. Antioxidants, glycosides, steroids, carbohydrates, prostaglandins, saponins, essential oil, phenols, and terpenoids were also detected. The methanolic fruit extract showed the presence of prostaglandins, antioxidants, carbohydrates, steroids, mycotoxin, phenols, saponins, terpenes, glycosides, and essential oil ( Figure 3).  The methanolic fruit extract showed the presence of prostaglandins, antioxidants, carbohydrates, steroids, mycotoxin, phenols, saponins, terpenes, glycosides, and essential oil ( Figure 3). water at 100 °C indicated the occurrence of flavonoids and the primary/secondary amines. Antioxidants, glycosides, steroids, carbohydrates, prostaglandins, saponins, essential oil, phenols, and terpenoids were also detected. The methanolic fruit extract showed the presence of prostaglandins, antioxidants, carbohydrates, steroids, mycotoxin, phenols, saponins, terpenes, glycosides, and essential oil ( Figure 3).    Table 1 lists the various phytochemicals identified in different A. mexicana extracts prepared in diverse solvents at room temperature and high temperature. + denotes the intensity of the chemical ingredients present in the extracts. Increase in the number of (+) indicates an increase in the intensity. ND-not detected.
The leaf extract prepared in these solvents contained terpenoids. The ethyl acetate extract contained cardiac glycosides, phenols, and traces of flavonoids. The aqueous and methanolic leaf extracts contained carbohydrates, phenols, quinones, and traces of flavonoids.
The stem extract of A. mexicana contained only terpenoids, flavonoids, quinones, and small amounts of steroids. Carbohydrates, cardiac glycosides, flavonoids, phenols, quinones, steroids, and terpenoids were detected in the ethyl acetate extract made at a low temperature. The methanolic extract produced at room temperature contained high amounts of alkaloids, carbohydrates, flavonoids, phenols, quinones, saponins, steroids, and terpenoids. The aqueous extract prepared under this condition contained only carbohydrates, phenols, and quinones.
The fruit extract obtained in hexane at a low temperature contained cardiac glycosides, quinones, terpenoids, and phenols. The ethyl acetate extract contained cardiac glycosides, steroids and terpenoids, phenols, and quinones, with traces of carbohydrates. The methanolic fruit extract contained alkaloids, carbohydrates, cardiac glycosides, flavonoids, quinones, steroids, terpenoids, and traces of phenol, whereas the aqueous extract contained carbohydrates, flavonoids, terpenoids, tannins, phenol, and quinones. The flower extract obtained in various solvents at a low temperature did not have significant phytochemicals (unpublished data).

Qualitative Analysis of the Phytochemicals Present in Different A. mexicana Extracts Prepared at a High Temperature
The leaf extract in hexane obtained at a high temperature contained terpenoids, quinones, and alkaloids, with carbohydrates and cardiac glycosides in small amounts. The ethyl acetate leaf extract contained cardiac glycosides, terpenoids and small amounts of phenols and flavonoids. The leaves extracted in methanol at a high temperature contained high amounts of alkaloids, carbohydrates, phenols, terpenoids, and tannins. Similar phytochemicals were found in the aqueous leaf extract made at 100 • C, except for small amounts of quinones.
The stem extracted in hexane at a high temperature contained carbohydrates, cardiac glycosides, quinones, and terpenoids in significant amounts, followed by phenols and traces of flavonoids. The ethyl acetate extract contained cardiac glycosides, terpenoids, quinones, phenols, and small amounts of flavonoids. Methanol and aqueous stem extracts prepared at a high temperature contained significant amounts of alkaloids, carbohydrates, phenols, terpenoids, and tannins. The aqueous extract also contained quinones.
The fruit extract prepared in hexane contained cardiac glycosides, quinones, steroids, terpenoids, and small amounts of flavonoids and phenols. The ethyl acetate extract contained cardiac glycosides, steroids, and terpenoids, as well as small amounts of carbohydrates, phenols, and quinones. Methanolic and aqueous fruit extracts prepared at a high temperature contained significant amounts of carbohydrates, phenols, quinones, and terpenoids. Steroids were present in methanolic extracts, but absent in aqueous extracts. The aqueous extracts, but not methanolic extracts, contained alkaloids and tannins.
Only the flower extracts prepared using methanol and water had detectable phytochemicals. The methanolic flower extract obtained at a high temperature contained significant amounts of carbohydrates, phenols, quinones, alkaloids, cardiac glycosides, terpenoids, and traces of flavonoids. The aqueous extract contained carbohydrates, phenols, quinones, tannins, and terpenoids, as well as small amounts of alkaloids and flavonoids (Table 1).

Free-Radical Neutralizing Ability of A. mexicana Extracts
The leaf extract revealed the highest free-radical scavenging activity in water, followed by methanol and ethyl acetate at room temperature and the temperature equivalent to their boiling points. The free-radical neutralizing ability of stem extracts in various solvents were as follows: methanol > aqueous > ethyl acetate in extracts prepared at low temperatures. The free-radical quenching activity of the stem extracts obtained at high temperatures was in the following order: ethyl acetate > water > methanol. The fruit extract made at room temperature exhibited similar activity in methanol and water. The fruit extract made at a temperature equivalent to their boiling point revealed similar activity in water and methanol, which was less than that in ethyl acetate. The flower extract displayed free-radical scavenging activity only when made at a temperature equivalent to the boiling points in methanol and water. The IC 50 values are listed in Table 2.

Effect of A. mexicana Extract against the Activity of HIV-1 RT
The leaf extract prepared in distilled water at a high temperature exhibited the maximum inhibitory effect against HIV-1 RT, with an IC 50 value of 0.044 mg/mL, followed by ethyl acetate and methanolic extracts. Only the leaf extract prepared in the ethyl acetate at room temperature displayed HIV-1 RT activity (Table 3). Similarly, only the ethyl acetate fruit extract prepared at high and low temperatures exhibited HIV-1 RT activity. No activity of anti-HIV-RT was detected in other extracts.

Antimicrobial Activity of Various A. mexicana Extracts
The antimicrobial effects of different extracts prepared at room temperature against different bacterial strains are listed in Table 4. Ampicillin (5 mg/mL) was used as a standard. The ethyl acetate and methanolic leaf extracts prepared at a low temperature were effective against S. typhi, Citrobacter, Shigella flexineri, and S. epidermis. Neisseria gonorrhoeae was inhibited only by the ethyl acetate leaf extract synthesized at room temperature. The ethyl acetate and methanolic fruit extracts prepared at room temperature inhibited Salmonela typhi, Neisseria gonorrhoeae, and S. flexineri. S. epidermis was inhibited only by the methanolic fruit extract.   Table 5 presents the antimicrobial effects of the plant extracts synthesized at the temperature equivalent to the boiling points of the solvents. Methanol and aqueous leaf extracts prepared at high temperatures inhibited Gonococci and Citrobacter. Salmonella typhi was inhibited by aqueous and ethyl acetate extracts, and Staphylococcus epidermis was inhibited only by the ethyl acetate leaf extract. Methanolic and ethyl acetate fruit extracts prepared at a high temperature inhibited Salmonella typhi and Citrobacter. Only the methanolic fruit extract inhibited Gonococci and S. flexineri, whereas S. epidermis was inhibited only by the ethyl acetate fruit extract.  Methanol

Discussion
A. mexicana belongs to the Papaveraceae family, which possesses antibacterial [18][19][20], antifungal [21,22], antiviral [23,24], antioxidant [25,26], and cytotoxic/anticancer [27] properties. The emergence of drug-resistant microbial strains [28] has necessitated the search for antimicrobial compounds from natural sources, such as plants and marine microorganisms. Many studies have determined the antimicrobial effects and secondary metabolites of A. mexicana [29,30]. Recently, A study by Orozco-Nunnelly et al., 2021 [28] has shown that the methanolic extract of outer roots and leaves displayed strong antimicrobial activities, especially against Gram-positive bacteria. In this study, different extracts of A. mexicana have been shown tocause an inhibition in the growth of both Gram +ve and Gram −ve bacteria. The results from the present study revealed that the temperatures corresponding to the solvents' boiling points resulted in a better yield of phytochemicals. Terpenoids were the most abundant, followed by phenols, flavonoids, carbohydrates, and cardiac glycosides, as also reported by [29]. The leaf and the stem extracts exhibited higher freeradical scavenging activity than the fruit and flower extracts, which may be attributed to phenolic compounds [31]. The leaf extract possessed antioxidant activity, as also shown by Perumal et al. (2010) [32].
HIV uses reverse transcriptase (RT) to convert its RNA to cDNA. Ishizuka et al., 2020 [23] have shown that RT exhibits RNA-and DNA-dependent DNA polymerase activities and RNase H activity. In the present study, the aqueous extract of leaves prepared at a high temperature indicated the maximum inhibitory activity against HIV-1 RT, much like any other nonnucleoside reverse transcriptase inhibitor (NNRTI), as demonstrated by Sanna et al., 2018 [33]. The fruit extracts prepared at a high temperature also exhibited anti-HIV-RT activity, which may be attributed to the presence of flavonoids and alkaloids in these extracts [34]. Nuciferine, an alkaloid in the roots of A. mexicana, has been reported to be responsible for its anti-HIV-1RT activity [35]. The methanolic extracts of the entire A. mexicana contains benzo[c]phenanthridine alkaloid and (±)-6-acetonyl dihydrochelerythrine, which exhibited anti-HIV activity [35]. The anti-HIV-RT property was found to be mainly associated with the aqueous extract of the leaves at a high temperature. The results from earlier studies in collaboration with other works have shown anti-HIV-RT activity by some organic compounds present in the leaf extracts, displaying an inhibition of HIV-1 DNA polymerase function [23]. Various plant extracts exhibited antimicrobial activities against different test organisms, namely S. typhi, Gonococci, Citrobacter, S. flexineri, and S. epidermis. Among the extracts prepared at room temperatures, the methanolic extract revealed the best antimicrobial activity. On the other hand, among the extracts prepared at high temperatures, the aqueous extract revealed the maximum antimicrobial activity. The antimicrobial activity may be attributed to phenols, terpenoids, and alkaloids [36].
The methanolic extracts of the outer root of A. mexicana possessed maximum activity against Gram+ve bacteria, and minimum activity against the Gram−ve bacteria and fungi (Orozco-Nunnelly et al., 2021) [28]. The antimicrobial activity of the leaf extract can be attributed to the presence of berberine, an alkaloid [28]. Berberine exerts its effect by damaging the cell membrane and impeding the synthesis of proteins and DNA [37]. Berberin chloride, in combination with various anti-staphylococcal drugs as a reference of CoNS strains, varied greatly across different bacterial stains and drugsused [38]. Rahman et al., 2011 [19] reported that the leaf extract of A. mexicana inhibited Salmonella sp, which was also noted in this study.

Reagents and Chemicals
The reagents Luria-Bertani broth (Miller, Appleton, WI, USA) and Mueller-Hinton agar were procured from SRL Pvt. Ltd. (Mumbai, India), Himedia, whereas the ampicillin sodium injection was obtained from Saralife (New Delhi, India). Silica-gel-coated TLC plates, Wagner's reagent, and Molisch reagent were procured from Merck, Darmstadt, Germany. All other reagents used were of analytical grade.

Plant Material Collection and Extract Preparation
A. mexicana plants were collected from Prayagraj, India, and nearby areas. The leaves, stems, flowers, and fruits were collected. The collected plant materials were washed under running tap water and then with double-distilled water. The plant parts were then dried in shade and powdered. The powder was dissolved in the ethyl acetate, hexane, water, and methanol at a low temperature (room temperature) and high temperature (temperatures equivalent to the boiling points of the solvents such as 77.1 • C, 68 • C, 100 • C, and 64.7 • C, respectively). The extracts at a high temperature were prepared using the Soxhlet apparatus. These extracts were dried at room temperature and used by dissolving them into the desired volume of the solvents for using them at different concentrations for carrying out different specific experiments, or stored at −20 • C until further use.

Analysis of A. mexicana Extract Spectrophotometrically
The spectrophotometric analysis of the extracts resulted in different spectral patterns corresponding to the specific absorption profiles under UV and visible wavelengths of various components present in the isolated extracts determined through spectrophotometry. The UV-visible spectra of different concentrations of extracts were placed in a quartz cuvette and recorded from 190 to 750 nm by using the double-beam UV-visible spectrophotometer (model: Scientific Spectroscan UV2700, Thermo Fisher Scientific India Pvt. Ltd., Pune, India) [39,40]. The analysis of the absorption peaks was carried out and compared with the peaks of specific bioactive compounds.

Thin Layer Chromatography (TLC)
The TLC plates coated with the silica gel (Merck, Darmstadt, Germany) were employed for the analysis of different phytochemicals present in the various extracts of A. mexicana. The fractions of which the UV-visible absorption spectra showed the presence of optimal concentrations of phytochemicals were subject to thin layer chromatography to confirm the results.

2,2-Diphenyl-1-picrylhydrazyl Assay
The free radical quenching potential of various extracts was determined using the 2, 2-diphenyl-1-picrylhydrazyl assay (DPPH), as described previously [43], with some modifications. Of the 10 mM DPPH solution, 1 mL was added to different extract concentrations (25,50,100,200, 300, and 500 µg/mL). After 30 min incubation at room temperature, the absorbance was determined at 517 nm. The ascorbic acid (1%) was used as a standard. The free radical scavenging capacity of the extracts was calculated as shown below:

Examination of Different Plant Extracts for Their Anti-HIV-1RT Activity
The anti-HIV-1RT activity of different plant extracts was determined by employing RT colorimetric assay kits (Roche, NY, USA), in accordance with the manufacturer's instruction.

Test Microorganisms
Clinically pathogenic bacteria, namely Neisseria gonorrhoeae, Salmonella typhi, Citrobacter, Staphylococcus epidermidis, and Shigella flexineri, were used. These organisms were obtained from the Medical Sciences Institute of BHU, Varanasi, India.

Antibacterial Activity
The agar-well diffusion method [44] was used to examine antibacterial susceptibility. Sterilized discs (6 mm) soaked in different extract concentrations (100, 200, 300, and 500 µg in 5 µL volume each) of extracts were placed on solid media with sterile forceps. Water and ampicillin (5 µg/mL) were employed as the negative and positive controls, respectively. The petri plates were placed for 24 h at 37 • C. The diameter of the zone of inhibition (mm) after 24 h incubation was used as a measure of antimicrobial activity.

Statistical Analysis
We performed all experiments in triplicate. Data are presented as the mean ± SE of the three replicates. All the data were statistically analyzed using (MS Excel)-Microsoft 365. (Graph pad Prism)-GraphPad Software, Inc., San Diego, CA, USA.

Conclusions
In the present study, the leaf extract of A. mexicana prepared in an aqueous medium at high temperatures was found to be the most active against HIV 1-RT and the tested microorganisms. Water at a high temperature was the best solvent to extract the pharmacological activities of bioactive molecules from A. mexicana. The pharmacological properties of A. mexicana may be attributable to various compounds, which may exert their actions through specific and nonspecific mechanisms. Therefore, active chemical components should be identified to elucidate their mechanisms of actions for the scientific utilization of the plant to cure various diseases.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28114428/s1, Figure S1: Spectrophotometric analysis of the phytochemicals present in various parts of A. mexicana, extracts prepared at room temperature (24 ± 2 • C) in various solvents. A-hexane, B-ethyl acetate, C-methanol, and D-water; Figure S2: Spectrophotometric analysis of the phytochemicals present in various parts of A. mexicana, extracts prepared at high temperature in various solvents (corresponding to their boiling points). A-hexane, B-ethyl acetate, C-methanol, and D-water.; Table S1: Spectrophotometric analysis of A. mexicana